Direct Gene Transfer Research Articles

Direct gene transfer of adenoviral-mediated interferon α into human bladder cancer cells but not the bystander factors produced induces endoplasmic reticulum stress-related cytotoxicity

We have previously shown that adenoviral-mediated interferon α (Ad-IFNα) is cytotoxic both to cells that are sensitive to recombinant interferon α (IFNα) and to cells which are resistant to IFNα. The cancer cell-specific cytotoxic effects of Ad-IFNα involve three different mechanisms: 1. The direct effect of IFNα production causing cancer cell death in IFNα sensitive cells (1); 2. The direct effect of Ad-IFNα infection and high levels of IFNα expression in IFNα resistant cancer cells (2); and 3. The indirect effect of the Ad-IFNα bystander factors produced (2-4). After Ad-IFNα infection, the cells produce a large amount of perinuclear localized IFN protein. This protein over-load could be a major factor in the direct cancer cell death of those cells infected with Ad-IFNα compared with the indirect cytotoxic effects of the bystander factors produced. Here, we investigated whether a component of Ad-IFN-induced cell death involves protein overload-induced endoplasmic reticulum (ER) stress, using an IFNα-resistant human bladder cancer cell line (KU7), and the normal human urothelial cell line, TERT-NHUC, as preclinical models. We found that the two ER stress response pathways examined were activated in KU7 cells. In contrast, following treatment of the normal TERT-NHUC cells with Ad-IFNα, no ER stress signals were observed. In addition, no ER stress related changes were seen, when KU7 cells were exposed to conditioned medium from Ad-IFNα-treated KU7 cells, indicating that bystander produced cytotoxicity did not involve ER stress. After 24 h of Ad-IFNα infection, the KU7 cancer cells produced spliced X-box binding protein 1 and activating transcription factor 6 protein (ATF6), evoking an ER stress response that could contribute to Ad-IFNα induced apoptosis in these cancer cells. In addition, GADD153/CHOP, GADD34 and BAX were also subsequently modified following activation of the ER stress pathways, thereby signaling downstream effectors in a pro-apoptotic manner.

The vesicular glutamate transporter-1 upstream promoter and first intron each support glutamatergic-specific expression in rat postrhinal cortex

Multiple applications of direct gene transfer into neurons require restricting expression to glutamatergic neurons, or specific subclasses of glutamatergic neurons. Thus, it is desirable to develop and analyze promoters that support glutamatergic-specific expression. The three vesicular glutamate transporters (VGLUTs) are found in different populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported on a plasmid (amplicon) Herpes Simplex Virus vector that contains a VGLUT1 promoter. This vector supports long-term expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex, but does not support expression in VGLUT2-containing glutamatergic neurons in the ventral medial hypothalamus. This VGLUT1 promoter contains both the VGLUT1 upstream promoter and the VGLUT1 first intron. In this study, we begin to isolate and analyze the glutamatergic-specific regulatory elements in this VGLUT1 promoter. We show that the VGLUT1 upstream promoter and first intron each support glutamatergic-specific expression. We isolated a small, basal VGLUT1 promoter that does not support glutamatergic-specific expression. Next, we fused either the VGLUT1 upstream promoter or the first intron to this basal promoter. The VGLUT1 upstream promoter or the first intron, fused to the basal promoter, each supported glutamatergic-specific expression in POR cortex.

Direct gene transfer in the Gottingen minipig CNS using stereotaxic lentiviral microinjections.

We aim to induce direct viral mediated gene transfer in the substantia nigra (SN) of the Gottingen minipig using MRI guided stereotaxic injections of lentiviral vectors encoding enhanced green fluorescent protein (EGFP). Nine female Gottingen minipigs were injected unilaterally into the SN with 6 per 2.5 microliters lentivirus capable of transducing cells and mediating expression of recombinant EGFP. The animals were euthanized after four (n=3) or twenty weeks (n=6). Fresh brain tissue from three animals was used for PCR. The remaining six brains were cryo- or paraffin-sectioned for fluorescence, Nissl-, and immunohistochemical EGFP visualization. EGFP was seen in nigral neurons, axons, glial cells, endothelial cells, and in nigral fibers targeting the striatum. PCR-based detection confirmed the presence of the transgene in SN, whereas all other examined brain areas were negative, indicating that the immunohistochemically detected EGFP in the striatum derived from transfected nigral cells.

Nontransgenic genome modification in plant cells.

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.

Identified circuit in rat postrhinal cortex encodes essential information for performing specific visual shape discriminations.

Learning theories hypothesize specific circuits encode essential information for performance. For simple tasks in invertebrates and mammals, the essential circuits are known, but for cognitive functions, the essential circuits remain unidentified. Here, we show that some essential information for performing a choice task is encoded in a specific circuit in a neocortical area. Rat postrhinal (POR) cortex is required for visual shape discriminations, protein kinase C (PKC) pathways mediate changes in neuronal physiology that support learning, and specific PKC genes are required for multiple learning tasks. We used direct gene transfer of a constitutively active PKC to prime a specific POR cortex circuit for learning visual shape discriminations. In the experiment, rats learned a discrimination, received gene transfer, learned new discriminations, received a small lesion that ablated approximately 21% of POR cortex surrounding the gene transfer site, and were tested for performance for discriminations learned either before or after gene transfer. Lesions of the genetically targeted circuit selectively interfered with performance for discriminations learned after gene transfer. Activity-dependent gene imaging confirmed increased activity in the genetically targeted circuit during learning and showed the essential information was sparse-coded in approximately 500 neurons in the lesioned area. Wild-type rats contained circuits with similar increases in activity during learning, but these circuits were located at unpredictable, different positions in POR cortex. These results establish that some essential information for performing specific visual discriminations can be encoded in a small, identified, neocortical circuit and provide a foundation for characterizing the circuit and essential information.

Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C–Staphylococcus A protein

Because of the heterogeneous cellular composition of the brain, and especially the forebrain, cell type-specific expression will benefit many potential applications of direct gene transfer. The two prevalent approaches for achieving cell type-specific expression are using a cell type-specific promoter or targeting gene transfer to a specific cell type. Targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors modifies glycoprotein C (gC) to replace the heparin binding domain, which binds to many cell types, with a binding activity for a specific cell surface protein. We previously reported targeted gene transfer to nigrostriatal neurons using chimeric gC–glial cell line-derived neurotrophic factor or gC–brain-derived neurotrophic factor protein. Unfortunately, this approach is limited to cells that express the cognate receptor for either neurotrophic factor. Thus, a general strategy for targeting gene transfer to many different types of neurons is desirable. Antibody-mediated targeted gene transfer has been developed for targeting specific virus vectors to specific peripheral cell types; a specific vector particle protein is modified to contain the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. Here, we report antibody-mediated targeted gene transfer of HSV-1 vectors to a specific type of forebrain neuron. We constructed a chimeric gC–ZZ protein, and showed this protein is incorporated into vector particles and binds Ig G. Complexes of these vector particles and an antibody to the NMDA receptor NR1 subunit supported targeted gene transfer to NR1-containing neocortical neurons in the rat brain, with long-term (2months) expression.

Low Current-driven Micro-electroporation Allows Efficient In Vivo Delivery of Nonviral DNA into the Adult Mouse Brain

Viral gene transfer or transgenic animals are commonly used technologies to alter gene expression in the adult brain, although these approaches lack spatial specificity and are time consuming. We delivered plasmid DNA locally into the brain of adult C57BL/6 mice in vivo by voltage- and current-controlled electroporation. The low current-controlled delivery of unipolar square wave pulses of 125 µA with microstimulation electrodes at the injection site gave 16 times higher transfection rates than a voltage-controlled electroporation protocol with plate electrodes resulting in currents of about 400 mA. Transfection was restricted to the target region and no damage due to the electric pulses was found. Our current-controlled electroporation protocol indicated that the use of very low currents resulting in applied voltages within the physiological range of the membrane potential, allows efficient transfection of nonviral plasmid DNA. In conclusion, low current-controlled electroporation is an excellent approach for electroporation in the adult brain, i.e., gene function can be influenced locally at a high level with no mortality and minimal tissue damage.

Microprojectile bombardment mediated genetic transformation of embryo axes and plant regeneration in cumin (Cuminum cyminum L.)

Pre-cultured cumin embryos were bombarded under 27 inches Hg vacuum, 25 mm distance from rupture disc to macrocarrier, 10 mm macrocarrier flight distance using 1100 psi rupture disc and 9 cm microprojectile travel distance. An average of 110 embryos was used per shot and 91% embryos showed transient GUS expression after 24 h. Shoot tips and roots of T0 plantlets exhibited GUS expression done after 3 months of bombardment. Transformation was confirmed with PCR amplification of 0.96 and 1.3 kb band of hptII and gus genes respectively from T0 transgenics and southern blot analysis using PCR amplified DIG labeled hptII gene as probe. It is the first successful attempt of transformation of cumin plant through direct gene transfer using particle gene gun and adequately exhibiting the possibility of stable transformation in cumin.

A helper virus-free HSV-1 vector containing the vesicular glutamate transporter-1 promoter supports expression preferentially in VGLUT1-containing glutamatergic neurons

Multiple potential uses of direct gene transfer into neurons require restricting expression to specific classes of glutamatergic neurons. Thus, it is desirable to develop vectors containing glutamatergic class-specific promoters. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported a plasmid (amplicon) Herpes Simplex Virus (HSV-1) vector that placed the Lac Z gene under the regulation of the VGLUT1 promoter (pVGLUT1lac). Using helper virus-free vector stocks, we showed that this vector supported ∼ 90% glutamatergic neuron-specific expression in postrhinal (POR) cortex, in rats sacrificed at either 4 days or 2 months after gene transfer. We now show that pVGLUT1lac supports expression preferentially in VGLUT1-containing glutamatergic neurons. pVGLUT1lac vector stock was injected into either POR cortex, which contains primarily VGLUT1-containing glutamatergic neurons, or into the ventral medial hypothalamus (VMH), which contains predominantly VGLUT2-containing glutamatergic neurons. Rats were sacrificed at 4 days after gene transfer, and the types of cells expressing ß-galactosidase were determined by immunofluorescent costaining. Cell counts showed that pVGLUT1lac supported expression in ∼ 10-fold more cells in POR cortex than in the VMH, whereas a control vector supported expression in similar numbers of cells in these two areas. Further, in POR cortex, pVGLUT1lac supported expression predominately in VGLUT1-containing neurons, and, in the VMH, pVGLUT1lac showed an ∼ 10-fold preference for the rare VGLUT1-containing neurons. VGLUT1-specific expression may benefit specific experiments on learning or specific gene therapy approaches, particularly in the neocortex.

English

  Chickpea production is limited worldwide because of abiotic and biotic stresses. Efforts to overcome these production constraints through traditional breeding are difficult due to limited genetic variation. Novel regeneration is pre-requisite for genetic transformation offers the opportunity to overcome hybridization barriers and introduce novel genes for resistance. Although direct gene transfer via direct DNA transfer has been reported, Agrobacterium mediated transformation is the preferred method and standard protocols have been established for the production of transgenic plantlets derived from co-cultivation of embryonic axes. This was soon adopted due to difficulties associated with regeneration of whole plants from callus. Only few reports have been reported using genetic transformation/ transgene(s) against abiotic stress tolerance transgenic chickpea plants. Transgenic chickpea using bacterial codA gene tolerance against abiotic stresses have been developed. Chickpea improvement and application of genomics tools to the study of the chickpea genome will be enhanced through the use of genetic transformation.   Key words: Cicer arietinum L., TDZ, Indole butyric acid, (IBA), efficient rooting, aeration, transplantation, transformation.

Arterial spin labeling CMR quantifies increased perfusion in hearts of mice treated with cardioprotective, AAV9-mediated EcSOD gene therapy prior to myocardial infarction

Introduction Experimental myocardial infarction (MI), direct gene transfer with adeno-associated viral (AAV) vectors and CMR in mice are powerful tools for studying the roles of individual genes in MI and post-MI left ventricular (LV) remodeling. Arterial spin labeling (ASL) enables the quantification of myocardial perfusion (MP) by CMR, but is sensitive to variable heart rates and irregular respiration, prohibiting accurate measurement early after MI. We developed a cardio-respiratory gated (CRG) ASL method that is insensitive to these factors to measure MP in mice.

Systematic Evaluation of AAV Vectors for Liver directed Gene Transfer in Murine Models

Vectors based on adeno-associated viruses (AAVs) are being evaluated for use in liver-directed gene therapy. Candidates have been preselected on the basis of capsid structure that plays an important role in determining performance profiles. We describe a comprehensive and statistically powered set of mouse studies designed to compare the performance of vectors based on seven novel AAV capsids. The key criteria used to select candidates for successful gene therapy are high level and stable transgene expression in the absence of toxicity. Based on these criteria, the best performing vectors, AAV8, AAVhu.37, and AAVrh.8, will be further evaluated in nonhuman primates (NHPs).

Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages

Directed gene transfer into specific cell lineages in vivo is an attractive approach for both modulating gene expression and correcting inherited mutations such as emphysema caused by human alpha1 antitrypsin (hAAT) deficiency. However, somatic tissues are mainly comprised of heterogeneous, differentiated cell lineages that can be short lived and difficult to specifically transfect. Here, we describe an intratracheally instilled lentiviral system able to deliver genes selectively to as many as 70% of alveolar macrophages (AMs) in the mouse lung. Following a single in vivo lentiviral transduction, genetically tagged AMs persisted in lung alveoli and expressed transferred genes for the lifetime of the adult mouse. A prolonged macrophage lifespan, rather than precursor cell proliferation, accounted for the surprisingly sustained presence of transduced AMs. We utilized this long-lived population to achieve localized secretion of therapeutic levels of hAAT protein in lung epithelial lining fluid. In an established mouse model of emphysema, lentivirally delivered hAAT ameliorated the progression of emphysema, as evidenced by attenuation of increased lung compliance and alveolar size. After 24 weeks of sustained gene expression, no humoral or cellular immune responses to hAAT protein were detected. Our results challenge the dogma that AMs are short lived and suggest that these differentiated cells may be a possible target cell population for in vivo gene therapy applications, including the sustained correction of hAAT deficiency.

Flexibility in cell targeting by pseudotyping lentiviral vectors.

Lentiviral vectors have become an important research tool and have just entered into clinical trials. As wild-type lentiviruses engage specific receptors that have limited tropism, most investigators have replaced the endogenous envelope glycoprotein with an alternative envelope. Such pseudotyped vectors have the potential to infect a wide variety of cell types and species. Alternatively, selection of certain viral envelope glycoproteins may also facilitate cell targeting to enhance directed gene transfer. We describe the method for generating pseudotyped vector and provide information regarding available pseudotypes and their respective target tissues.

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium × hortorum, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium x hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-microF capacitor in a 250-V cm(-1) electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33-36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 microS cm(-1) allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l(-1) kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l(-1) kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.

Progress and Prospects: prospects of repeated pulmonary administration of viral vectors

Pulmonary gene therapy may ultimately cure diseases such as cystic fibrosis, alpha1-antitrypsin deficiency, lung cancer and pulmonary hypertension. Efficient expression of delivered genes in target cell types is essential for the achievement of this goal. To this end, re-administration of viral vectors may be required (1) to increase the percentage of transduced airway epithelial cells, (2) to direct gene transfer to individual lobes during successive delivery sessions or (3) to boost attenuated expression over time. Immune responses to viral proteins or viral-encoded proteins are the greatest barrier to repeated vector administration.

0279: Optimization of Ultrasound-Targeted Microbubble Destruction Mediated Gene Transfection in Vitro

Ultrasound-targeted microbubble destruction (UTMD) has evolved as a new, promising tool for site-specific drug and gene delivery. However, the transgenic efficiency, cell viability should be optimized and the integrity of plasmid DNA must be ensured not to be affected. This study was designed to perform the systematic optimization on ultrasound direct gene transfer to cells and establish the optimal parameters of UTMD facilitating gene transfection in vitro.

Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.

Local Gene Transfer and Expression Following Intramuscular Administration of FGF-1 Plasmid DNA in Patients With Critical Limb Ischemia

NV1FGF is an expression plasmid encoding sp.FGF-1(21-154) currently under investigation for therapeutic angiogenesis in clinical trials. NV1FGF plasmid distribution and transgene expression following intramuscular (IM) injection in patients is unknown. The study involved six patients with chronic critical limb ischemia (CLI) planned to undergo amputation. A total dose of 0.5, 2, or 4 mg NV1FGF was administered as eight IM injections (0.006, 0.25, or 0.5 mg per injection) 3-5 days before amputation. Injected sites (30 cm(3)) were divided into equally sized smaller pieces to assess spatial distribution of NV1FGF sequences (PCR), NV1FGF mRNA (reverse transcriptase-PCR), and fibroblast growth factor-1 (FGF-1)-expressing cells (immunohistochemistry). Data indicated gene expression at all doses. The distribution area was within 5-12 cm for NV1FGF sequences containing the expression cassette, up to 5 cm for NV1FGF mRNA, and up to 3 cm for FGF-1-expressing myofibers. All FGF receptors were detected indicating robust potential for bioactivity after NV1FGF gene transfer. Circulating levels of NV1FGF sequences were shown to decrease within days after injection. Data support demonstration of plasmid-mediated gene transfer and expression in muscles from patients with CLI. FGF-1 expression was shown to be limited to injection sites, which supports the concept of multiple-site injection for therapeutic use.

Erythropoiesis Stimulating Agents and Techniques: A Challenge for Doping Analysts

Recombinant human erythropoietin (rHuEPO) engineered in Chinese hamster ovary (CHO) cell cultures (Epoetin alfa and Epoetin beta) and its hyperglycosylated analogue Darbepoetin alfa are known to be misused by athletes. The drugs can be detected by isoelectric focusing (IEF) and immunoblotting of urine samples, because "EPO" is in reality a mixture of isoforms and the N-glycans of the recombinant products differ from those of the endogenous hormone. However, there is a plethora of novel erythropoiesis stimulating agents (ESAs). Since the originator Epoetins alfa and beta are no longer protected by patent in the European Union, rHuEPO biosimilars have entered the market. In addition, several companies in Asia, Africa and Latin America produce copied rHuEPOs for clinical purposes. While the amino acid sequence of all Epoetins is identical, the structure of their glycans differs depending on the mode of production. Some products contain more acidic and others more basic EPO isoforms. Epoetin delta is special, as it was engineered by homologous recombination in human fibrosarcoma cells (HT-1080), thus lacking N-glycolylneuraminic acid like native human EPO. ESAs under development include EPO fusion proteins, synthetic erythropoiesis stimulating protein (SEP) and peptidic (Hematide(), CNTO 528) as well as non-peptidic EPO mimetics. Furthermore, preclinical respectively clinical trials have been performed with small orally active drugs that stimulate endogenous EPO production by activating the EPO promoter ("GATA-inhibitors": diazepane derivatives) or enhancer ("HIF-stabilizers": 2-oxoglutarate analogues). The prohibited direct EPO gene transfer may become a problem in sports only in the future.